MiRNome variations in milk fractions during feed restrictions of different intensities in dairy cows.

BACKGROUND: In dairy cows, diet is one factor that can affect their milk production and composition. However, the effect of feed restriction on milk miRNome has not yet been described. Indeed, milk is the body fluid with the highest RNA concentration, which includes numerous microRNA. Its presence in the four different milk fractions, whole milk, fat globules, mammary epithelial cells and extracellular vesicles, is still poorly documented. This study aimed to describe the effects of different feed restrictions on the miRNome composition of different milk fractions. RESULTS: Two feed restrictions were applied to lactating dairy cows, one of high intensity and one of moderate intensity. 2,896 mature microRNA were identified in the different milk fractions studied, including 1,493 that were already known in the bovine species. Among the 1,096 microRNA that were sufficiently abundant to be informative, the abundance of 1,027 of them varied between fractions: 36 of those were exclusive to one milk fraction. Feed restriction affected the abundance of 155 microRNA, with whole milk and milk extracellular vesicles being the most affected, whereas milk fat globules and exfoliated mammary epithelial cells were little or not affected at all. The high intensity feed restriction led to more microRNA variations in milk than moderate restriction. The target prediction of known microRNA that varied under feed restriction suggested the modification of some key pathways for lactation related to milk fat and protein metabolisms, cell cycle, and stress responses. CONCLUSIONS: This study highlighted that the miRNome of each milk fraction is specific, with mostly the same microRNA composition but with variations in abundance between fractions. These specific miRNomes were affected differently by feed restrictions, the intensity of which appeared to be a major factor modulating milk miRNomes. These findings offer opportunities for future research on the use of milk miRNA as biomarkers of energy status in dairy cows, which is affected by feed restrictions.

Deep RNA-Seq reveals miRNome differences in mammary tissue of lactating Holstein and Montbéliarde cows.

BACKGROUND: Genetic polymorphisms are known to influence milk production and composition. However, the genomic mechanisms involved in the genetic regulation of milk component synthesis are not completely understood. MicroRNAs (miRNAs) regulate gene expression. Previous research suggests that the high developmental potential of the mammary gland may depend in part on a specific miRNA expression pattern. The objective of the present study was to compare the mammary gland miRNomes of two dairy cow breeds, Holstein and Montbéliarde, which have different mammogenic potentials that are related to differences in dairy performance. RESULTS: Milk, fat, protein, and lactose yields were lower in Montbéliarde cows than in Holstein cows. We detected 754 distinct miRNAs in the mammary glands of Holstein (n = 5) and Montbéliarde (n = 6) midlactating cows using RNA-Seq technology, among which 738 were known and 16 were predicted miRNAs. The 25 most abundant miRNAs accounted for 90.6% of the total reads. The comparison of their abundances in the mammary glands of Holstein versus Montbéliarde cows identified 22 differentially expressed miRNAs (Padj ≤ 0.05). Among them, 11 presented a fold change ≥2, and 2 (miR-100 and miR-146b) were highly expressed. Among the most abundant miRNAs, miR-186 is known to inhibit cell proliferation and epithelial-to-mesenchymal transition. Data mining showed that 17 differentially expressed miRNAs with more than 20 reads were involved in the regulation of mammary gland plasticity. Several of them may potentially target mRNAs involved in signaling pathways (such as mTOR) and lipid metabolism, thereby indicating that they could influence milk composition. CONCLUSION: We found differences in the mammary gland miRNomes of two dairy cattle breeds. These differences suggest a potential role for miRNAs in mammary gland plasticity and milk component synthesis, both of which are related to milk production and composition. Further research is warranted on the genetic regulation of miRNAs and their role in milk synthesis.

DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part II - the impact of lactation rank.

Mastitis is among the main reasons women cease breastfeeding. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, the effect of inflammation on the mammary gland is not completely understood. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation after in vivo intramammary challenges and the differences in DNA methylation between 1st and 2nd lactations. Lactation rank induces 981 differential methylations of cytosines (DMCs) in mammary tissue. Inflammation in 1st lactation compared to inflammation in 2nd lactation results in the identification of 964 DMCs. When comparing inflammation in 1st vs. 2nd lactations with previous inflammation history, 2590 DMCs were identified. Moreover, Fluidigm PCR data show changes in the expression of several genes related to mammary function, epigenetic regulation, and the immune response. We show that the epigenetic regulation of two successive physiological lactations is not the same in terms of DNA methylation and that the effect of lactation rank on DNA methylation is stronger than that of the onset of inflammation. The conditions presented here show that few DMCs are shared between comparisons, suggesting a specific epigenetic response depending on lactation rank, the presence of inflammation, and even whether the cells had previously suffered inflammation. In the long term, this information could lead to a better understanding of the epigenetic regulation of lactation in both physiological and pathological conditions.Abbreviations: RRBS, reduced representation bisulphite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; MaSC, mammary stem cell; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process.

DNA methylation and gene expression changes in mouse mammary tissue during successive lactations: part I - the impact of inflammation.

Mastitis is among the main reasons women cease breastfeeding, which leads to them supplementing breast milk with artificial formula. In farm animals, mastitis results in significant economic losses and the premature culling of some animals. Nevertheless, researchers do not know enough about the effect of inflammation on the mammary gland. This article discusses the changes to DNA methylation in mouse mammary tissue caused by lipopolysaccharide-induced inflammation (4 h post-injection of lipopolysaccharide). We analysed the expression of some genes related to mammary gland function, epigenetic regulation, and the immune response. The analysis focused on three comparisons: inflammation during the first lactation, inflammation during second lactation with no history of inflammation, and inflammation during second lactation with previous inflammation. We identified differentially methylated cytosines (DMCs), differentially methylated regions (DMRs), and some differentially expressed genes (DEGs) for each comparison. The three comparisons shared some DEGs; however, few DMCs and only one DMR were shared. These observations suggest that inflammation is one of several factors affecting epigenetic regulation during successive lactations. Furthermore, the comparison between animals in second lactation with and without inflammation, with no inflammation history during first lactation showed a different pattern compared to the other conditions in this experiment. This indicates that inflammation history plays an important role in determining epigenetic changes. The data presented in this study suggest that lactation rank and previous inflammation history are equally important when explaining mammary tissue gene expression and DNA methylation changes.Abbreviations: RRBS, reduced representation bisulfite sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; MEC, mammary epithelial cells; TSS, transcription start site; TTS, transcription termination site; UTR, untranslated region; SINE, short interspersed nuclear element; LINE, long interspersed nuclear element; CGI, CpG island; DEG, differentially expressed gene; DMC, differentially methylated cytosine; DMR, differentially methylated region; GO term, gene ontology term; MF, molecular function; BP, biological process.

Milk proteins as a feed restriction signature indicating the metabolic adaptation of dairy cows.

Milk production in dairy cows is affected by numerous factors, including diet. Feed restriction is known to have little impact on milk total protein content but its effect on the fine protein composition is still poorly documented. The objective of this study was to describe the effects of two feed restriction trials of different intensities on the milk protein composition of Holstein cows. One restriction trial was of high intensity (H: 8 mid-lactation Holstein cows) and the second of moderate intensity (M: 19 peak lactation Holstein cows). Feed restriction decreased the milk protein yield for caseins under the M trial and of all six major milk proteins under the H trial. These decreased yields lead to lower concentrations of αs1-, αs2- and ß-caseins during the H trial. The milk proteome, analyzed on 32 milk samples, was affected as a function of restriction intensity. Among the 345 proteins identified eight varied under the M trial and 160 under the H trial. Ontology analyses revealed their implication in carbohydrate, lipid and protein metabolisms as well as in the immune system. These proteins reflected adaptations of the animal and mammary gland physiology to feed restriction and constituted a signature of this change.

Short communication: Mouse mammary tumor virus driven α-lactalbumin expression effects on lactation and fertility of transgenic mice.

α-Lactalbumin (Alac) is one of the major milk proteins. Its gene expression is restricted to epithelial cells of the lactating mammary gland. The Alac interaction with a uridine 5'-diphosphate-galactosyltransferase induces lactose synthesis, a major osmotic regulator of milk secretion. Other functions attributed to this protein include induction of apoptosis and anti-inflammatory activities. To assess if forced expression of this gene during early gestation or involution could affect mammary physiology, an Alac-encoding minigene was expressed in transgenic mice under the transcriptional regulation of the mouse mammary tumor virus promoter. The mammary expression did not interfere with gestation, resulted in a slight increase in milk yield as indirectly assessed by the 11% increased growth rate of the pups reared by transgenic females compared with that of those reared by control mice, and induced a slight delay in the early involution process, as demonstrated by histological analyses. The use of the mouse mammary tumor virus promoter resulted in Alac expression in several nonmammary tissues, such as the brain, the testis, the ovary, and the uterus. Although it did not affect male reproductive performances, it induced a female subfertile phenotype, characterized by embryonic implantation failure in the transgenic female reproductive tract.

Characterization of Holstein and Normande whole milk miRNomes highlights breed specificities.

The concept of milk as a healthy food has opened the way for studies on milk components, from nutrients to microRNAs, molecules with broad regulatory properties present in large quantities in milk. Characterization of these components has been performed in several species, such as humans and bovine, depending on the stages of lactation. Here, we have studied the variation in milk microRNA composition according to genetic background. Using high throughput sequencing, we have characterized and compared the milk miRNomes of Holstein and Normande cattle, dairy breeds with distinct milk production features, in order to highlight microRNAs that are essential for regulation of the lactation process. In Holstein and Normande milk, 2,038 and 2,030 microRNAs were identified, respectively, with 1,771 common microRNAs, of which 1,049 were annotated and 722 were predicted. The comparison of the milk miRNomes of two breeds allowed to highlight 182 microRNAs displaying significant differences in the abundance. They are involved in the regulation of lipid metabolism and mammary morphogenesis and development, which affects lactation. Our results provide new insights into the regulation of molecular mechanisms involved in milk production.

Rat prolactin synthesis by lactating mammary epithelial cells.

It has previously been suggested that the mammary cell could produce prolactin (PRL). This hypothesis was investigated by incubation with [35S]methionine-cysteine followed by SDS-PAGE, immunoblotting and autoradiography of immunoprecipitated PRL, and by electron microscopic analysis after incubation without or with cycloheximide. Immunoreactive 14-, 23-, 25-, 32- and 36-kDa PRL forms were radioactive. By two-dimensional electrophoresis analysis, immunoreactive and radioactive spots, of about 25 kDa and high molecular weight, were also detected. After incubation of mammary epithelial cells with cycloheximide, immunogold electron microscopy showed a drastic decrease of labelling in organelles involved in synthesis and secretion, compared to those incubated in control medium. These results make it possible to conclude that lactating mammary tissue is able to synthesize PRL.

Goat PRND expression pattern suggests its involvement in early sex differentiation.

Expression of the goat prion protein gene locus was assessed by reverse transcriptase-polymerase chain reaction on testes and ovaries at various developmental stages. A weak and stochastic expression of the PRNP and PRNT genes was observed. For PRNT, it is consistent with the detected deletions of two single nucleotides within its open reading frame in ruminant genes. PRND was expressed in both tissues at all stages. Whereas its expression is constant in the ovaries, it increases in testes between 36 and 46 days postcoitum (dpc) and remains high thereafter. In testes, Doppel was found in the nucleus of germinal cells and in the cytoplasm of Leydig cells at 44 dpc. It was detected in the cytoplasm of Leydig cells and of some Sertoli and germinal cells at 62 dpc. In the ovaries, it was observed in the nucleus of germinal cells at 44 dpc and mainly in their cytoplasm at 62 dpc. This expression pattern was shown to parallel that of C-kit and suggests Doppel involvement in early testis differentiation.

A cDNA macroarray resource for gene expression profiling in ruminant tissues involved in reproduction and production (milk and beef) traits.

cDNA arrays have proven to be useful tools to screen gene expression in many animal species including livestock species. A collaborative program was launched to construct a ruminant cDNA collection, representative of three tissues: Muscle, Embryo and Mammary gland, named MEM. This collection gathers clones mainly arising from 3 non-normalised cDNA libraries: a directed bovine muscle library, a 14-day-old bovine embryo library and a goat lactating mammary library. It is made up of 1896 clones (637 muscle, 882 embryo and 377 mammary cDNAs), selected after sequencing and bioinformatic analyses. Amplification products yielded from these clones as well as controls were printed onto Nylon membranes to generate macroarrays. Hybridisation with relevant cDNA targets allowed checking the location of about 50 cDNAs and the specificity of each sub-set of the repertoire. Macroarrays were hybridised with radiolabelled cDNA complex targets from five different tissues (muscle, embryo, mammary gland, adipose tissue and oocyte). Both somatic and germinal complex targets gave valid hybridisation signals with 45 to 80% of the printed probes. This specific cDNA collection now provides a powerful tool for transcriptomic studies with the ultimate objective to better understand physiological and metabolic functions in ruminants. It will be subsequently included into a forthcoming larger collection.

Characterization of the goat lactoferrin cDNA: assignment of the relevant locus to bovine U12 synteny group.

Lactoferrin (LTF), which is the major iron-binding protein in milk and physiological fluids, belongs to the transferrin family. We report here the sequence of a caprine LTF cDNA, 2411 bp in length, encoding the pre-protein (709 amino acid residues). Sequence comparisons reveal that structural features, including iron-binding sites, cysteine residues involved in disulphide bonds are remarkably conserved between LTF proteins from various species. Of the 5 potential glycosylation sites identified, only one site appears to be conserved between artiodactyls, rodents and humans. Using a somatic cell hybrid panel, the LTF locus was assigned to the bovine U12 syntenic group. This assignment and the localization of the LTF gene on bovine chromosome 22 (BTA 22) by Schwerin et al. (1) using fluorescent in situ hybridization achieves an additional analogy between a synteny group and a chromosome in cattle. Since serum transferrin (STF) had been previously mapped on BTA 1, in cattle LTF and STF loci are not localized on the same chromosome, conversely to the situation observed in humans (HSA 3) and mice (MMU 9).

A survey of the goat genome transcribed in the lactating mammary gland.

To fulfill its primary function, which is to synthesize milk during the course of lactation, the mammary gland requires efficient transcriptional, translational, and secretory machineries involving multiple genes among which promising candidates underlying the genetic variation of milk production have to be found. With the aim of providing a first transcriptional profile of lactating mammary tissue, a non-normalized cDNA library has been constructed from the udder of a lactating goat. After having discarded cDNA clones encoding the major milk proteins the rapid characterization of genes expressed in this tissue, by automated partial cDNA sequencing, was used to analyze a total of 435 cDNA clones. Examination of the Expressed Sequence Tags (ESTs) for similarities with sequence databases identified 234 cDNAs corresponding to 140 unique genes or proteins. Eighty-three clones, not similar to any current database entries, representing 77 novel sequences unrelated to previously described genes, were thus identified. Tissue specificity and relative abundance of 18 of these 77 unidentified clones were examined by dot blot and RT-PCR experiments. Sequence data were subsequently used to assign six genes of unknown localization in the bovine genome, to synteny groups by use of bovine-hamster cell hybrids and PCR.

Cytogenetic mapping of 25 goat mammary gland Expressed Sequence Tags (ESTs).

Today, there is a shift towards a positional candidate approach in the molecular identification of genes. This study reports on an Expressed Sequence Tags (ESTs) mapping initiative in goats, based on sequence information gathered from a previous mammary gland cDNA systematic sequencing project. A total of 25 novel genes was localised cytogenetically on 16 goat chromosomes. Six of these ESTs were found to map to cattle milk QTL regions. These results made it possible to assess the use of ESTs as a shortcut to the molecular identification of some QTLs and as a valuable tool for comparative mapping.

Prolactin gene expression in ovine and caprine mammary gland.

The presence of prolactin (PRL) mRNA in the mammary gland of lactating goats and sheep was demonstrated by Northern analysis and RT-PCR. This provides evidence that the PRL gene is transcribed in this tissue. This ectopic expression is not restricted to the lactational period, as PRL transcripts were also found during the last third of pregnancy. Comparison of mammary and pituitary PRL mRNAs showed that they are similar in size but less abundant in mammary gland. In addition, an 847-bp cDNA fragment amplified from mammary retrotranscripts, containing the entire coding region and the major part of the 5' and 3' untranslated regions (UTRs), was found to be identical in sequence to its pituitary counterpart. Primer extension analysis, performed to obtain further information on the structure of the mammary PRL mRNA, has shown that the 5' UTR is 56 nucleotides (nt) long for both species. This is comparable with the size (53 nt) found using the caprine pituitary RNA as template. These results strongly suggest that the PRL gene is not transcribed from a different promoter in mammary gland, as has been demonstrated for placental and lymphocyte cells, but is more likely transcribed from the pituitary-specific promoter. Finally, the presence of PRL mRNA in polysomal fractions suggests that PRL is synthesized in mammary cells.

[Use of progestagens in subcutaneous implants for the control of sexual cycles in the cow]. / Utilisation de progestagènes en implants sous-cutanés pour la maîtrise des cycles sexuels chez les bovins

PIP: Estrus synchronization and related fertility were studied in cattle using subcutaneous implants containing norethandrolone, SC 21009, or fluorogestone acetate. Lengthening the duration of treatment decreases the rate of estrus inhibition. For long-term (16-18 days) treatments, the percentage of cows showing estrus with 4 days from implant removal increases with the initial content of the implant; 61.5% and 84.6% with 6 and 12 mg SC21009 respectively. The progestagen content of the implant has little effect on fertility, but longer duration of treatment lowers fertility: 49.4% and 27.3% for 10 and 16 days, respectively, for SC 21009 implants. Fluorogestone acetate had little effect. Estradiol valerate injection at the time of implant insertion increases fertility regardless of treatment duration. The use of high-potency progestagens permits the use of small implants. Cross-link percentage must be studied for each progestagen. Of the progestagens studied, only SC 21009 is sufficiently potent for implant use.^ieng